Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome characterized by reduced erythropoiesis and increased cancer risk. Congential mutations in ribosomal protein genes (RPGs) are most closely linked with this syndrome. It is unclear why decreased RPG expression selectively decreases erythropoiesis and predisposes to cancers that typically require increased protein synthesis necessary for rapid growth. Our recent data may provide a molecular connection between reduced RPG expression and the clinical phenotypes observed in DBA. A high-throughput screen to discover effectors and regulators of human microRNA (miRNA) function identified RPGs as a novel class of genes regulating miRNA activity. Specifically, knockdown of every RPG decreased miRNA activity and thus increased expression of miRNA-target mRNAs. In this application, we similarly propose that in DBA, RPG mutations reduce miRNA activity which alters expression of developmentally-regulated genes and oncogenes (both enriched in miRNA target sites), leading to reduced erythropoiesis and increased cancer risk. To test this mechanism of decreased erythropoiesis in DBA, we will obtain DBA patient and normal donor bone marrow and tissue samples. We will generate induced pluripotent stem (iPS) cells from both sources. We will compare erythropoiesis from bone marrows and iPS from normal donors with and without RPG knockdown to bone marrows and iPS from DBA patients. We will characterize and isolate discreet erythroid progenitors from each of these sources. We will then isolate total RNA, monosomal RNA, and polysomal RNA from each progenitor and perform miRNA (Luminex bead) and mRNA (RNA-seq) expression profiling. Transcriptome and translatome maps will be generated and compared to each other and to miRNA expression profiles from matched control samples. We will use these data sets as inputs for unbiased gene network analyses to identify affected (miRNA-regulated) pathways when RPG expression is reduced. We will then confirm the roles of genes in the predicted networks by manipulating their expression in hopes of reversing erythropoiesis phenotypes in RPG knockdown and DBA patient samples. We will use siRNAs or specific miRNA mimics to knockdown genes whose mRNA levels or translation robustly increased when RPG expression was reduced. Conversely, we will use cDNAs to reconstitute genes whose total mRNA levels or translation robustly decreased when RPG expression was reduced. Our studies will establish novel connections between RPG expression, miRNA function, miRNA-targeted gene expression, and erythropoiesis, and may reveal new targets for DBA therapy.